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Binding proteins of MTMR9
Holšteinová, Aneta ; Doubravská, Lenka (advisor) ; Cebecauer, Marek (referee)
Myotubularins are lipid phosphatases that dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate the position three of the inositol ring. This allows them to regulate the structure of the lipid layer of the membrane compartment. The first member of the family was described in association with a severe hereditary myopathy. From that point on, another thirteen members have been added to the family. The catalytically inactive MTMR9 carrying the conserved mutation in the phosphatase domain regulates the localization of the marker of the early secretory pathway, RAB1A, the cis-Golgi structure and the secretion. MTMR9 interacts with the catalytically active MTMR6 and MTMR8 that specifically localizes and increases their phophatase activity. The aim of this diploma thesis was to find out whether the phenotype observed in cells with altered MTMR9 levels is dependent on the catalytically active phosphatases MTMR6 and MTMR8. We proved the influence of MTMR6 and MTMR8 on the distribution of tranfected RAB1A between the intermediate compartment and the Golgi apparatus. MTMR6 and MTMR8 also take part in regulating the cis-Golgi structure. By the use of two different approaches we did not manage to clarify the influence of MTMR6 and MTMR8 on secretion. Changes in the catalytic...
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Regulation of LAT trafficking to the plasma membrane
Rakhimbekova, Anastasia ; Cebecauer, Marek (advisor) ; Černý, Jan (referee)
Linker for activation of T cells is a palmitoylated transmembrane adaptor protein, which is expressed in most of immune cells, but not in immature and mature B cells. It plays an important role in T-cell activation and maturation. LAT is synthesized in the endoplasmic reticulum. Its sorting to the plasma membrane is controlled with various determinants, such as properties of the transmembrane domain and structural motifs in the intracellular part of the protein. Some of those determinants are important for posttranslational modifications, export from the Golgi apparatus and, probably, membrane microdomain targeting, while others interact with COPII machinery and mediate protein export from the endoplasmic reticulum or targeting to endosomes. Keywords: LAT, functional motifs, protein sorting, plasma membrane, Golgi apparatus
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Cloning and characterisation of selected Class II formins
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Rösel, Daniel (referee)
Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...
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